Biology subjects

Kumar, N. G.

Publications and source records attributed to Kumar, N. G..

10 recordsLinked to original sources

bioRxiv2025

TRPV1 Defends the Healthy Murine Cornea against Staphylococcus aureus Adhesion Independently of Sensory Nerve Firing

PurposePreviously we showed that transient receptor potential ion channels TRPA1 and TRPV1 selectively protect the cornea against bacterial adhesion, with TRPA1 countering the Gram-negative pathogen Pseudomonas aeruginosa and TRPV1 countering environmental bacteria. Here, we explored parameters of this specificity using a Gram-positive pathogen Staphylococcus aureus. MethodsHealthy corneas of C57BL/6J wild-type (WT), TRPA1 (-/-) or TRPV1 (-/-) mice were challenged with S. aureus for 4 or 6 hours. Some experiments instead/also used resiniferatoxin (RTX) to deplete TRPV1-expressing nerves, JNJ-17203212 to selectively antagonize TRPV1, or the anesthetic bupivacaine to inhibit nerve firing. Adherent bacteria were quantified using FISH labeling (16S rRNA-targeted probe). Lyz2+, CD11c+ and CD45+ cells were visualized/quantified using hybrid mT/mG + LysMcre mice (red cell membranes; Lyz2+-GFP), CD11c+-YFP mice, and anti-CD45-antibody respectively. ResultsCorneas of TRPV1 (-/-) not TRPA1 (-/-) mice were found more susceptible to S. aureus adhesion compared to WT. Accordingly, either ablation of TRPV1-expressing nerves or TRPV1 antagonism increased adhesion. Defense against S. aureus adhesion did not depend on nerve firing. Despite having no significant impact on CD11c+ or Lyz2+ cell numbers, S. aureus challenge increased CD45+ cell counts, also dependent on TRPV1-expressing nerves, and it increased Lyz2+ cell sphericity and volume. ConclusionHealthy corneas utilize TRPV1 to protect against S. aureus adhesion independently of sensory nerve firing. This contrasts with defense against P. aeruginosa adhesion which requires TRPA1 and nerve firing. How the differential immune cell responses to these two pathogens relate to TRP-dependent defense against adhesion remains to be determined.

microbiology↗

bioRxiv2025

Cross-membrane cooperation among bacteria can facilitate intracellular pathogenesis

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to cause life- and sight-threating infections. Once considered an extracellular pathogen, numerous studies have shown it can survive intracellularly. Previously, we showed that P. aeruginosa inside cells can diversify into distinct subpopulations in vacuoles and the cytoplasm. Here, we report that the transition from vacuoles to cytoplasm requires collaboration with the extracellular subpopulation, through Ca2+ influx enabled by their type III secretion system (T3SS) translocon pore proteins. Moreover, we show that collaboration among P. aeruginosa subpopulations can contribute to disseminating intracellular bacteria in vivo in a mouse infection model. This study provides the basis for future studies to investigate how cooperation of extracellular and intracellular bacteria within the host may contribute to disease progression and persistence.

microbiology↗

bioRxiv2025

Exploring Synergies in Brain-Machine Interfaces: Compression vs. Performance

Individuals with severe neurological injuries often rely on assistive technologies, but current methods have limitations in accurately decoding multi-degree-of-freedom (DoF) movements. Intracortical brain-machine interfaces (iBMIs) use neural signals to provide a more natural control method, but currently struggle with higher-DoF movements--something the brain handles effortlessly. It has been theorized that the brain simplifies high-DoF movement through muscle synergies, which link multiple muscles to function as a single unit. These synergies have been studied using dimensionality reduction techniques like principal component analysis (PCA), non-negative matrix factorization (NMF), and demixed PCA (dPCA) and successfully used to reduce noise and improve offline decoder stability in non-invasive applications. However, their effectiveness in improving decoding and generalizability for implanted recordings across varied tasks is unclear. Here, we evaluated if brain and muscle synergies can enhance iBMI performance in non-human primates performing a two-DoF finger task. Specifically, we tested if PCA, dPCA, and NMF could compress and denoise brain and muscle data and improve decoder generalization across tasks. Our results showed that while all methods effectively compressed data with minimal loss in decoding accuracy, none improved performance through denoising. Additionally, none of the methods enhanced generalization across tasks. These findings suggest that while dimensionality reduction can aid data compression, alone it may not reveal the "true" control space needed to improve decoder performance or generalizability. Further research is required to determine whether synergies are the optimal control framework or if alternative approaches are required to enhance decoder robustness in iBMI applications. Significance StatementMany researchers believe that brain and muscle synergies represent a fundamental control strategy and could enhance brain-machine interface (BMI) decoding performance. These synergies, extracted through dimensionality reduction techniques, are thought to simplify complex neural data, improving the efficiency and accuracy of BMI systems. In our study, we evaluated brain and muscle synergies in a dexterous finger task. We found that while these synergies effectively compressed high-dimensional data, they did not improve performance through denoising or generalize well across different contexts. Instead, the highest performance was achieved when using all available data, suggesting that synergies, although useful for data compression, may not provide the "true" control space needed to enhance decoder robustness or adaptability in implanted BMI systems.

neuroscience↗

bioRxiv2025

The Pseudomonas aeruginosa T3SS can contribute to traversal of an in situ epithelial multilayer independently of the T3SS needle

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a {Delta}pscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a {Delta}exsD mutant constitutively on for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes ({Delta}pscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted ({Delta}pscU-L{Delta}exoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented {Delta}pscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to development of therapeutics targeting the T3SS system. IO_SCPLOWMPORTANCEC_SCPLOWWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of mechanisms involved is derived largely from cell culture experimentation. The latter is due to practical limitations of in vivo/in situ models and challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by: 1) leveraging the transparent properties and superficial location of the cornea, 2) using our established method for enabling bacterial traversal susceptibility, and 3) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

microbiology↗

bioRxiv2024

Contact Lens Wear Alters Transcriptional Responses to Pseudomonas aeruginosa in Both the Corneal Epithelium and the Bacteria

PurposeHealthy corneas resist colonization by virtually all microbes yet contact lens wear can predispose the cornea to sight-threatening infection with Pseudomonas aeruginosa. Here, we explored how lens wear changes corneal epithelium transcriptional responses to P. aeruginosa and its impact on bacterial gene expression. MethodsMale and female C57BL/6J mice were fitted with a contact lens on one eye for 24 h. After lens removal, corneas were immediately challenged for 4 h with P. aeruginosa. A separate group of naive mice were similarly challenged with bacteria. Bacteria-challenged eyes were compared to uninoculated naive controls as was lens wear alone. Total RNA-sequencing determined corneal epithelium and bacterial gene expression. ResultsPrior lens wear profoundly altered the corneal response to P. aeruginosa, including: upregulated pattern-recognition receptors (tlr3, nod1), downregulated lectin pathway of complement activation (masp1), amplified upregulation of tcf7, gpr55, ifi205, wfdc2 (immune defense) and further suppression of efemp1 (corneal stromal integrity). Without lens wear, P. aeruginosa upregulated mitochondrial and ubiquinone metabolism genes. Lens wear alone upregulated axl, grn, tcf7, gpr55 (immune defense) and downregulated Ca2+-dependent genes necab1, snx31 and npr3. P. aeruginosa exposure to prior lens wearing vs. naive corneas upregulated bacterial genes of virulence (popD), its regulation (rsmY, PA1226) and antimicrobial resistance (arnB, oprR). ConclusionPrior lens wear impacts corneal epithelium gene expression altering its responses to P. aeruginosa and how P. aeruginosa responds to it favoring virulence, survival and adaptation. Impacted genes and associated networks provide avenues for research to better understand infection pathogenesis.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa can diversify after host cell invasion to establish multiple intracellular niches

Within epithelial cells, Pseudomonas aeruginosa depends on its type three secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole associated-bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light and electron microscopy, we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant ({Delta}exsA) unable to escape vacuoles phenocopied vacuolar-associated sub-populations in wild-type PAO1-infected cells, results revealing that epithelial cell death depended upon bacterial viability. Intra-vital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular sub-populations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic sub-populations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. ImportancePseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative sub-population of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm, and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this sub-population, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing it diversifies into multiple populations with T3SS-negative members enabling persistence whilst rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

microbiology↗

bioRxiv2019

Detecting Direct Oral Anticoagulants in Trauma Patients using Liquid Chromatography-Mass Spectrometry: a Novel Approach to Medication Reconciliation

BackgroundAccurate medication reconciliation in trauma patients is essential but difficult. Currently there is no established clinical method of detecting direct oral anticoagulants (DOACs) in trauma patients. We hypothesized that a liquid chromatography-mass spectrometry (LCMS) based assay can be used to accurately detect DOACs in trauma patients upon hospital arrival.\n\nMethodsPlasma samples were collected from 356 patients who provided informed consent including-10 healthy controls, 19 known positive or negative controls and 327 trauma patients over 65 years of age who were evaluated at our large, urban Level 1 Trauma Center. The assay methodology was developed in healthy and known controls to detect apixaban, rivaroxaban and dabigatran using LCMS and then applied to 327 samples from trauma patients. Standard medication reconciliation processes in the electronic medical record documenting DOAC usage was compared with LCMS results to determine overall accuracy, sensitivity, specificity and positive and negative predictive values (PPV, NPV) of the assay.\n\nResultsOf 356 patients, 39 were on DOACs (10.96%): 21 were on Apixaban, 14 on rivaroxaban and 4 on dabigatran. The overall accuracy of the assay for detecting any DOAC was 98.60%, with a sensitivity of 94.87% and specificity of 99.06%, (PPV 92.50% and NPV 99.37%). The assay detected apixaban with a sensitivity of 90.48% and specificity of 99.11% (PPV 86.36% and NPV 99.40%). There were three false positive results and two false negative LCMS results for apixaban. Dabigatran and rivaroxaban were detected with 100% sensitivity and specificity.\n\nConclusionsThis LCMS-based assay was highly accurate in detecting DOACs in trauma patients. Further studies need to confirm the clinical efficacy of this LCMS assay and its value for medication reconciliation in trauma patients.\n\nStudy type: diagnostic test\n\nBasic Science paper: therefore does not require a level of evidence.

pharmacology and toxicology↗

bioRxiv2019

Metabolic Modulation Predicts Heart Failure Tests Performance

Cardiopulmonary testing (CPET) and biomarkers such as NT-proBNP, Galectin-3, and C-reactive protein (CRP) have shown great promise for characterizing the heart failure (HF) phenotypes. However, the underlying metabolic changes that cause, predict and accompany changes in these parameters are not well known. In depth knowledge of these metabolic changes has the ability to provide new insights towards the clinical management of HF as well as providing better biomarkers for the assessment of disease progression and measurement of success from clinical interventions. To address this knowledge gap, we undertook a deep metabolomic and lipidomic phenotyping of a cohort of HF patients and utilized Multiple Regression Analysis (MRA) to identify associations between the patients plasma metabolome and the lipidome to parameters of CPET and the above mentioned HF biomarkers (HFBio). A total of 49 subjects were submitted to CPET and HFBio evaluation with deep metabolomic and lipidomic profiling of the plasma. Stepwise MRA and Standard Least Squares methods were used to determine models of best fit. Metabolic Pathway Analysis was utilized to detect what metabolites were over-represented and significantly enriched, and Metabolite Set Enrichment Analysis was used to explore pre-existing biological knowledge from studies of human metabolism. The study cohort was predominantly males of African American decent, presenting a high frequency of diabetes, hyperlipidemia, and hypertension with a low mean left ventricular ejection fraction and a high left ventricular end-diastolic and end-systolic volume. VE/VCO2 and Peak VO2 (CV=0.07 and 0.08, respectively) showed the best metabolic predictive model for test performance, while CRP and NT-ProBNP (CV=0.31 and 0.29, respectively) were the least fitted models. Aminoacyl-tRNA and amino acid biosynthesis, amino acid metabolism, nitrogen metabolism, pantothenate and CoA biosynthesis, sphingolipid and glycerolipid metabolism, fatty acid biosynthesis, glutathione metabolism, and pentose phosphate pathway (PPP) were revealed to be the most relevant pathways. Principal related diseases were brain dysfunction and enzyme deficiencies associated with lactic acidosis. Considering the modulations in HF poor test performance, our results indicate an overall profile of oxidative stress, lactic acidosis, and metabolic syndrome coupled with mitochondria dysfunction. Overall, the insights resulting from this studys MRA coincides with what has previously been discussed in parts in existing literature thereby confirming the validity of our findings while at the same time thoroughly characterizing the metabolic underpinning of CPET and HFBio results.

biochemistry↗

bioRxiv2018

Risk stratification of allogeneic stem cell recipients with respect to the potential for development of GVHD via their pre-transplant plasma lipid and metabolic signature

The clinical outcome of allogeneic hematopoietic stem cell transplantation (SCT) is strongly influenced from the complications arising during the post-transplant immune restoration and has been well studied and described. However, the metabolic status of the recipient pre-transplant also has the potential to influence this outcome and has never been studied before and has the potential to enable risk stratification with respect to the development of transplant associated complications such as graft vs. host disease (GVHD). In order to better understand this aspect of transplant related complications we investigated the pre-transplantation metabolic signature to assess the possibility of pre-transplant risk stratification. This pilot study was composed of 14 patients undergoing myeloablative conditioning followed by either HLA matched related, unrelated donor, or autologous stem cell transplantation. Blood samples were taken prior to transplant and the plasma was comprehensively characterized with respect to its lipidome and metabolome via LCMS and GCMS. The results indicated a significantly pro-inflammatory metabolic profile in patients who eventually developed Graft vs. Host Disease (GVHD). The data revealed 5 potential pre-transplant biomarkers (1-monopalmitin, diacylglycerol (DG) 38:5, DG 38:6, 2-aminobutyric acid, and fatty acid (FA) 20:1) that demonstrated high sensitivity and specificity towards predicting post-transplant GVHD development. The predictive model developed demonstrated an estimated predictive accuracy of risk stratification of 100%, with an Area under the Curve of the ROC of 0.995 with 100%. The likelihood ratio of 1-monopalmitin (infinity), DG 38:5 (6.0) and DG 38:6 (6.0) also demonstrated that a patient with a positive test result for these biomarkers pre-transplant will likely have very high odds of developing GVHD post-transplant. Collectively the data demonstrates the possibility of using pre-transplant metabolic signature for risk stratification of SCT recipients with respect to development of GVHD.

biochemistry↗

bioRxiv2018

A Pre-transplant Blood-based Lipid Signature for Prediction of Antibody-mediated Rejection in Kidney Transplant Patients

There is a lack of biomarkers for pre-kidney transplant immune risk stratification to avoid over- or under-immunosuppression, despite substantial advances in kidney transplant management. Since the circulating lipidome is integrally involved in various inflammatory process and pathophysiology of several immune response, we hypothesized that the lipidome may provide biomarkers that are helpful in the prediction of kidney rejection. Serial plasma samples collected over 1-year post-kidney transplant from a prospective, observational cohort of 45 adult Kidney Transplant [antibody-mediated rejection (AMR)=16; stable controls (SC) =29] patients, were assayed for 210 unique lipid metabolites by quantitative mass spectrometry. A stepwise regularized linear discriminant analysis (RLDA) was used to generate models of predictors of rejection and multivariate statistics was used to identify metabolic group differences. The RLDA models include lipids as well as of calculated panel reactive antibody (cPRA) and presence of significant donor-specific antibody (DSA) at the time of transplant. Analysis of lipids on day of transplant (T1) samples revealed a 7-lipid classifier (lysophosphatidylethanolamine and phosphatidylcholine species) which discriminated between AMR and SC with a misclassification rate of 8.9% [AUC = 0.95 (95% CI = 0.84-0.98), R2 = 0.63]. A clinical model using cPRA and DSA was inferior and produced a misclassification rate of 15.6% [AUC = 0.82 (95% CI = 0.69-0.93), R2 = 0.41]. A stepwise combined model using 4 lipid classifiers and DSA improved the AUC further to 0.98 (95% CI = 0.89-1.0, R2 = 0.83) with a misclassification of only 2.2%. Specific classes of lipids were lower in AMR compared with SC. Serial analysis of SC patients demonstrated metabolic changes between T1 and 6 months (T2) post-transplant, but not between 6 and 12 (T3) months post-transplant. There were no overtime changes in AMR patients. Analysis of SC T1 vs AMR T3 (that at time of AMR) showed sustained decreased levels of lipids in AMR at the time of rejection. These findings suggest that lack of anti-inflammatory polyunsaturated phospholipids differentiate SC from AMR pre-transplant and at the time of rejection, and a composite model using a 4-lipid classifier along with DSA could be used for prediction of antibody-mediated rejection before transplant. HighlightsO_LIDespite significant advancements in kidney transplant treatment and intensive clinical follow-up monitoring, all rejection events are unlikely to be recognized at the beginning. As a result, efforts have been made to identify new biomarkers for kidney rejection detection. C_LIO_LIWhile lipids are known to be potent mediators of inflammation, pro-resolving processes, and other cell signaling cascades, lipidomics can be applied to identify reliable biomarkers to monitor disease severity and may also allow prediction of kidney rejection. C_LIO_LIOur lipidomic study shows lipid profile changes between antibody-mediated rejection group and stable control group as a function of different time point, pre and post-kidney transplantation. Furthermore, our study demonstrates that combining lipid and clinical parameters allow prediction of rejection on the day of the transplant. C_LIO_LIThese findings have the potential to change the present paradigm of pre and post-transplant monitoring and management of these patients by implementing an evidence-based risk stratification technique, resulting in a substantial improvement in kidney transplant success. C_LI

biochemistry↗